The overall goal of the experiments performed in this laboratory are to identify the mechanisms controlling the activity of the hypothalamus-pituitary-adrenal (HPA) axis in fetal sheep. Providing a more complete understanding of the activity of the HPA axis will be key to understanding fetal stress, homeostasis, and (in sheep and perhaps in other species) the control of parturition. In past years, we have investigated several of the physiological and endocrine mechanisms controlling the activity of the ovine fetal HPA axis. We have reported that estrogen potently stimulates the fetal HPA axis, that the major circulating form of estradiol is estradiol-3-sulfate, and that exogenous estradiol-3-sulfate effectively stimulates fetal HPA axis activity. We have hypothesized that estradiol-3-sulfate could be taken up by the fetal brain directly through one or more organic anion transporters (OAT's), transporters that are known to transport sulfoconjugated estrogens. We have also hypothesized that the estradiol-3- sulfate might be deconjugated prior to both uptake and action. The proposed experiments are designed to test these hypotheses mechanistically. Specifically, we propose three specific aims: 1) to elucidate the roles of Organic Anion Transporters (OAT's) and steroid sulfatase (STS) in the uptake of sulfoconjugated estrogens by the fetal brain; 2) to test OAT's as mediators of estradiol-3-sulfate action in the fetal HPA axis; and 4) to test the physiological roles of estrogen receptors in HPA Axis responses to estradiol-3-sulfate. To achieve these aims, we will perform experiments using a combination of in vivo, pharmacological, biochemical, and molecular techniques. Together, these techniques will allow us to quantify estradiol-3-sulfate secretion and clearance kinetics and brain uptake in vivo, and use specific blockers of OAT and STS activity to test the roles played by these transport and deconjugation systems in vivo. Using physiological, biochemical, and molecular techniques well established in this laboratory, we will also be able to test the roles of these systems in the HPA axis response to estradiol-3-sulfate. We anticipate that the results of these studies will lead to the design of pharmacologic strategies for manipulating fetal HPA activity in utero. PUBLIC HEALTH RELEVANCE: Fetal stress is a prevalent cause of fetal death and morbidity in utero. Nevertheless, little is known about the mechanisms controlling the fetal stress hormones and we lack safe and effective ways of manipulating fetal stress hormone secretion. This proposal is aimed at providing a novel understanding of fetal stress hormones and novel approaches towards manipulating the fetal stress response.